THE KINETICS OF SIMIAN-VIRUS 40-INDUCED PROGRESSION OF QUIESCENT CELLS INTO S-PHASE DEPEND ON 4 INDEPENDENT FUNCTIONS OF LARGE T-ANTIGEN

Microinjection of purified simian virus 40 large-T-antigen protein or DNA encoding T antigen into serum-starved cells stimulates them to re- enter the cell cycle and progress through G(1) into the S phase. Genet ic analysis of T antigen indicated that neither its Rb/p107-binding ac tivity nor its p53 binding activity is essential to induce DNA synthes is in CV1P cells. However, T antigens bearing missense mutations that inactivate either activity induced slower progression of the cells int o the S phase than did wild-type T antigen. Inactivation of both activ ities resulted in a T antigen essentially unable to induce DNA synthes is. Missense mutations in either the DNA-binding region or the N termi nus also impaired the ability of full-length T antigen to stimulate DN A synthesis in CV1P cells. The wild-type kinetics of cell cycle progre ssion were restored by genetic complementation after coinjection of pl asmid DNAs encoding different mutant T antigens or conjection of purif ied mutant T-antigen proteins, suggesting that the four mitogenic func tions of T antigen are independent. The maximal rate of induction of D NA synthesis in secondary primate cells and established rodent cell li nes required the same four functions of T antigen. A model to explain how four independent activities could cooperate to stimulate cell cycl e progression is presented.